The control of overt differentiation of mammary gland in vitro involves an interplay between polypeptide and steroid hormones. Insulin, hydrocortisone and prolactin are absolutely required for the expression of milk protein genes. Role of individual hormones is being studied. These studies have shown: 1) In the presence of glucocorticoid and prolactin insulin is absolutely required for the expression of milk protein genes. In the present study it was shown that the accumulation effect on the 25K rat casein mRNA does not reflect stabilization of the transcript by insulin. Rather, insulin is essential for its synthesis in the presence of glucocorticoid and prolactin. Thus, insulin can be considered as a developmental hormone in the mammary system. Neither fetal calf serum nor Multiplication stimulating activity (MSA) or epidermal growth factor (EGF) can substitute insulin effect on differentation, though these hormones can sustain mammary cell vability in culture. 2) There is absolute requirement of hydrocortisone for accumulation of these messages, specifically the accumulation of 42K casein mRNA in mammary tissue from adrenalectomized, virgin rat is almost 20x higher in the presence of exogenous hydrocortisone than in its absence. Accumulation of 25K casein mRNA is also totally dependent on the steroid.